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2.  Altered Expression of MicroRNA-203 in Rheumatoid Arthritis Synovial Fibroblasts and Its Role in Fibroblast Activation 
Arthritis and rheumatism  2011;63(2):373-381.
Objective
MicroRNA (miRNA) are recognized as important regulators of a variety of fundamental biologic processes. Previously, we described increased expression of miR-155 and miR-146a in rheumatoid arthritis (RA) and showed a repressive effect of miR-155 on matrix metalloproteinase (MMP) expression in RA synovial fibroblasts (RASFs). The present study was undertaken to examine alterations in expression of miR-203 in RASFs and analyze its role in fibroblast activation.
Methods
Differentially expressed miRNA in RASFs versus osteoarthritis synovial fibroblasts (OASFs) were identified by real-time polymerase chain reaction (PCR)–based screening of 260 individual miRNA. Transfection of miR-203 precursor was used to analyze the function of miR-203 in RASFs. Levels of interleukin-6 (IL-6) and MMPs were measured by real-time PCR and enzyme-linked immunosorbent assay. RASFs were stimulated with IL-1β, tumor necrosis factor α (TNFα), lipopolysaccharide (LPS), and 5-azacytidine (5-azaC). Activity of IκB kinase 2 was inhibited with SC-514.
Results
Expression of miR-203 was higher in RASFs than in OASFs or fibroblasts from healthy donors. Levels of miR-203 did not change upon stimulation with IL-1β, TNFα, or LPS; however, DNA demethylation with 5-azaC increased the expression of miR-203. Enforced expression of miR-203 led to significantly increased levels of MMP-1 and IL-6. Induction of IL-6 by miR-203 overexpression was inhibited by blocking of the NF-κB pathway. Basal expression levels of IL-6 correlated with basal expression levels of miR-203.
Conclusion
The current results demonstrate methylation-dependent regulation of miR-203 expression in RASFs. Importantly, they also show that elevated levels of miR-203 lead to increased secretion of MMP-1 and IL-6 via the NF-κB pathway and thereby contribute to the activated phenotype of synovial fibroblasts in RA.
doi:10.1002/art.30115
PMCID: PMC3116142  PMID: 21279994
3.  Imbalance in distribution of functional autologous regulatory T cells in rheumatoid arthritis 
Annals of the Rheumatic Diseases  2007;66(9):1151-1156.
Objectives
Regulatory T cells (Tregs) exert their anti‐inflammatory activity predominantly by cell contact‐dependent mechanisms. A study was undertaken to investigate the regulatory capacity of autologous peripheral blood Tregs in contact with synovial tissue cell cultures, and to evaluate their presence in peripheral blood, synovial tissue and synovial fluid of patients with rheumatoid arthritis (RA).
Methods
44 patients with RA and 5 with osteoarthritis were included in the study. The frequency of interferon (IFN)γ‐secreting cells was quantified in synovial tissue cell cultures, CD3‐depleted synovial tissue cell cultures, synovial tissue cultures co‐cultured with autologous CD4+ and with CD4+CD25+ peripheral blood T cells by ELISPOT. Total CD3+, Th1 polarised and Tregs were quantified by real‐time PCR for CD3ε, T‐bet and FoxP3 mRNA, and by immunohistochemistry for FoxP3 protein.
Results
RA synovial tissue cell cultures exhibited spontaneous expression of IFNγ which was abrogated by depletion of CD3+ T cells and specifically reduced by co‐culture with autologous peripheral blood Treg. The presence of Treg in RA synovitis was indicated by FoxP3 mRNA expression and confirmed by immunohistochemistry. The amount of FoxP3 transcripts, however, was lower in the synovial membrane than in peripheral blood or synovial fluid. The T‐bet/FoxP3 ratio correlated with both a higher grade of synovial tissue lymphocyte infiltration and higher disease activity.
Conclusion
This study has shown, for the first time in human RA, the efficacy of autologous Tregs in reducing the inflammatory activity of synovial tissue cell cultures ex vivo, while in the synovium FoxP3+ Tregs of patients with RA are reduced compared with peripheral blood and synovial fluid. This local imbalance of Th1 and Treg may be responsible for repeated rheumatic flares and thus will be of interest as a target for future treatments.
doi:10.1136/ard.2006.068320
PMCID: PMC1955165  PMID: 17392348
4.  RANTES and Chemotactic Activity in Synovial Fluids From Patients With Rheumatoid Arthritis and Osteoarthritis 
Mediators of Inflammation  2005;2005(6):343-348.
A massive accumulation of inflammatory cells in synovial tissues is a major pathological feature of rheumatoid arthritis (RA). Neutrophiles dominate synovial fluid while rheumatoid synovium is infiltrated with mononuclear cells. Mechanisms regulating influx of particular subpopulations of leukocytes into articular cavity and synovium compartment are not completely defined. An increasing amount of data supports a crucial role of a C-C chemokine RANTES in the RA pathogenesis. Our objective is to evaluate chemotactic activity for neutrophils (NCA), lymphocytes (LCA), and monocytes (MoCA) in SFs obtained from patients with RA and osteoarthritis (OA). We also aimed to characterise the relation between chemotactic activity, RANTES, and percentage distribution of leukocytes in SF. SFs from 11 patients with RA and 6 with OA were included in the study. Modified microchamber Boyden method was employed to assess chemotactic activity. Cytological and biochemical analysis of SF was performed. RANTES was measured with ELISA. Rheumatoid SFs were rich in cells with predominance of neutrophiles while osteoarthritic fluids were lymphocytic. RA SFs were also characterised by increased lactoferrin level. Both NCA and LCA were higher in SF from patients with RA (62 ± 12 and 24 ± 6 cells/HPF, resp) as compared to patients with OA (23 ± 6; P < .05 and 6 ± 2 cells/HPF; P < 0.05). The chemoattractive effect of RA SF was more pronounced on neutrophiles than on lymphocytes. RA SF expressed high RANTES levels (145 ± 36 pg/mL), while OA SF was characterised by only trace amount of this chemokine (2 ± 1 pg/mL). We found positive correlation of RANTES with chemotactic activity for mononuclear cells (LCA+MoCA; R = 0.61; P < .05). Surprisingly, RANTES correlated also positively with neutrophiles number (R = 0.77; P < 0.001). Rheumatoid SF possesses strong chemotactic potency for leukocytes. RANTES is overexpressed in RA SF and is a potential mediator influencing intensity and composition of cellular infiltration in joints affected with inflammatory arthritis.
doi:10.1155/MI.2005.343
PMCID: PMC1533897  PMID: 16489254

Results 1-4 (4)